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OPEN
Received: 14 December 2016 Accepted: 10 August 2017 Published: xx xx xxxx
Secretory phospholipase A2IIA overexpressing mice exhibit cyclic alopecia mediated through aberrant hair shaft differentiation and impaired wound healing response Gopal L. Chovatiya1,2, Rahul M. Sarate1,2, Raghava R. Sunkara1,2, Nilesh P. Gawas1, Vineet Kala1 & Sanjeev K. Waghmare1,2 Secretory phospholipase A2 Group-IIA (sPLA2-IIA) is involved in lipid catabolism and growth promoting activity. sPLA2-IIA is deregulated in many pathological conditions including various cancers. Here, we have studied the role of sPLA2-IIA in the development of cyclic alopecia and wound healing response in relation to complete loss of hair follicle stem cells (HFSCs). Our data showed that overexpression of sPLA2-IIA in homozygous mice results in hyperproliferation and terminal epidermal differentiation followed by hair follicle cycle being halted at anagen like stage. In addition, sPLA2-IIA induced hyperproliferation leads to compl pathological conditions including various cancers. Here ete exhaustion of hair follicle stem cell pool at PD28 (Postnatal day). Importantly, sPLA2-IIA overexpression affects the hair shaft differentiation leading to development of cyclic alopecia. Molecular investigation study showed aberrant expression of Sox21, Msx2 and signalling modulators necessary for proper differentiation of inner root sheath (IRS) and hair shaft formation. Further, full-thickness skin wounding on dorsal skin of K14-sPLA2-IIA homozygous mice displayed impaired initial healing response. Our results showed the involvement of sPLA2-IIA in regulation of matrix cells differentiation, hair shaft formation and complete loss of HFSCs mediated impaired wound healing response. These novel functions of sPLA2-IIA may have clinical implications in alopecia, cancer development and ageing. Skin constantly renews itself throughout the adult life that acts as a protective barrier against pathogens, radiation etc.1. Adult skin is mainly composed of epidermis, dermis and hypodermis. Epidermal components mainly include interfollicular epidermis (IFE), hair follicle and sebaceous gland2. Dermis and hypodermis are mainly composed of collagen, elastic fibers and extrafibrillar matrix with various cell types including fibroblasts, adipose cells and macrophages. During embryogenesis, hair follicle is formed as an appendage of the epidermis by condensation of specialized mesenchymal cells (dermal papilla) in the dermis. The basal layer cells overlying mesenchymal cells get stimulated and subsequently form placode at E14.5 that proliferate and grow downward as a mature hair follicle at E16.5-E17.53. The hair follicle cycle comprises of distinct stages such as telogen (resting phase), anagen (growth phase) and catagen (regression phase)4. Initially, both the pulse-chase studies carried out using tritiated thymidine (3H) and BrdU showed the presence of infrequently dividing cells in the bulge of hair follicle5. Subsequently, pTre-H2BGFP/K5tTa (Tet off) double transgenic mice study showed that the hair follicle stem cells are highly dynamic, which divide infrequently and undergo random chromosome segregation to 1
Stem Cell Biology Group, Waghmare Lab, Cancer Research Institute, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, 410210, MH, India. 2Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai, 400085, India. Gopal L. Chovatiya and Rahul M. Sarate contributed equally to this work. Correspondence and requests for materials should be addressed to S.K.W. (email: [email protected])
SCIenTIfIC RepOrTS | 7: 11619 | DOI:10.1038/s41598-017-11830-9
1
www.nature.com/scientificreports/ maintain tissue homeostasis6–8. During follicle regeneration, the dermal papilla at the base of hair follicle provides initiatory signalling cues generated from the mesenchymal niche9. The cyclic growth of hair follicle is coordinated by the stem cells residing at the base of the bulge, which proliferate and migrate to provide progeny required for the hair-follicle regeneration and hair growth10. Further, the temporal activity of hair follicle stem cells is strictly dependent on interplay between mesenchymal niche and bulge, which is governed by secretion of various signalling modulators such as Wnt, BMP, Shh, and FGF11. In particular, Wnt signalling is necessary for the hair follicle morphogenesis and required for stem cell proliferation and differentiation during hair follicle regeneration12–14. Additionally, EGF induced EGFR signalling is indispensable for the initiation of hair growth15. The EGF signalling modulator, secretory phospholipase A2 group IIA (sPLA2-IIA) is also known as enhancing factor (EF), which expressed by paneth cells in the small intestine16, 17. sPLA2-IIA has two independent activities, catalytic and non-catalytic (enhancing) and both these activities rely on the two different domains of this enzyme18. Also, various studies have reported the expression of sPLA2-IIA in mouse epidermis19, 20. Moreover, sPLA2-IIA has been implicated in various forms of cancer such as intestinal, colorectal, prostrate21 and overexpression of sPLA2-IIA in mice epidermis showed increased susceptibility towards chemical carcinogenesis22. We have recently reported that sPLA2-IIA enhances the expression of Hb-EGF, EPGN and downstream c-Jun and Fos-B23. However, the molecular insight, if sPLA2-IIA regulates the hair shaft differentiation and the development of alopecia is still unknown. Secondly, do cells of other epidermal components form hair in the complete absence of hair follicle stem cells? Thirdly, whether wound healing process is impaired in the absence of HFSCs is yet to be determined. Notably, deregulation of various signalling modulators perturbs stem cells maintenance that may results in development and progression of cyclic alopecia. Impaired EGFR signalling by dominant negative mutant of epidermal growth factor receptor in the epidermis prevents the progression of the hair cycle to catagen stage and causes severe alopecia24. Further, knockout of Sox21 in mice alters differentiation of cuticle layer leading to development of cyclic alopecia25. Also, epidermal ablation of Smad4 resulted in hyperplasia of interfollicular epidermis (IFE) and sebaceous glands (SGs), that leads to exhaustion of the SC niche and progressive hair loss26. Expression of noggin in epidermis resulted in upregulated Wnt signalling with epidermal hyperplasia, progressive hair loss, and formation of trichofolliculoma-like tumors27. Msx2 deficiency exhibits abnormal structure of hair shafts and cycles of hair loss and regrowth28. Targeted disruption of Orai1 gene in mouse showed sporadic hair loss while inducible deletion of cnB1 gene in mice resulted in altered hair follicle structure and its mesenchyme adhesion, that causes cyclic alopecia29, 30. However, the molecular mechanism involved in cyclic alopecia is yet to be discovered. In this report, we studied the effect of K14-sPLA2-IIA expression in homozygous mice on alopecia and wound healing. To our knowledge, for the first time our findings on K14-sPLA2-IIA homozygous mice showed hair loss that is associated with an increased proliferation and differentiation of hair follicle stem cells, which led to exhaustion of hair follicle stem cells and development of cyclic alopecia at an early age. Additionally, K14-sPLA2-IIA homozygous mice showed impaired healing response during full thickness epidermal wounding.
Results
Altered hair follicle development and hair cycling in K14-sPLA2-IIA homozygous mice. To
check the expression pattern of sPLA2-IIA in mice epidermis, we performed the IHC staining of sPLA2-IIA on skin sections of wild type FVB mice at various postnatal ages. Our data showed that sPLA2-IIA expresses in basal layer, suprabasal layer and outer root sheath of hair follicle during morphogenesis, first hair cycle and one year old age (Supplementary Fig. S1). K14-sPLA2-IIA homozygous mice exhibited visible phenotypic growth abnormalities and are significantly smaller than the control littermates (Fig. 1a). Further, to confirm the nutritional status of the K14-sPLA2-IIA homozygous mice, we have quantified various nutritional parameters from the serum of the K14-sPLA2-IIA homozygous mice. Our data showed that there are no significant alterations in serum components such as serum albumin, Vitamin D 25-OH, Triglyceride, Sodium, Chloride, and marginal increase was observed in total protein, Vitamin B12, Calcium and Potassium (Supplementary Fig. S4). However, we have observed reduced serum glucose level after eight hours fasting in the K14-sPLA2-IIA homozygous mice (Supplementary Fig. S4). These results demonstrate that there are no significant alterations in the nutritional parameters of the K14-sPLA2-IIA homozygous mice. These K14-sPLA2-IIA homozygous mice showed progressive hair loss during hair follicle morphogenesis periods (PD15 and PD19). Further, haematoxylin and eosin staining (H&E) on the dorsal skin sections was performed during various postnatal days (PD15, 19, 21, 25, 28, 35, 41 and 49) (Fig. 1b). Our histological data revealed interfollicular epidermal cyst formation and abnormal thickening of the interfollicular epidermis (IFE) (Fig. 1c) as compared to wild type control littermate. Further, hair follicle cycling analysis on the dorsal skin at various postnatal days showed hair follicle is halted at anagen like stage in K14-sPLA2-IIA homozygous mice (Fig. 1d). This is due to the fact that we have not observed telogen at any postnatal day (PD15, PD17, PD21, PD25, PD28, PD30, PD35, PD41, PD45 and PD49). The change in morphology of the hair follicle is due to abnormal development of epidermal compartments. Further, we observed increased activation of β-catenin in K14-sPLA2-IIA homozygous mice skin as compared to hemizygous and wild type control littermate (Supplementary Fig. S2). These data indicate that the hair follicles of K14-sPLA2IIA homozygous mice failed to progress into regression phase (Catagen) of the hair follicle cycle. Thus, epidermal overexpression of sPLA2-IIA results in morphological abnormalities of hair follicle with hair loss.
Abnormal organization of epidermal components and stratification of epidermis.
Morphological abnormalities of the hair follicle led us to investigate if there is any effect on proliferation and differentiation in different epidermal compartments. To evaluate the effect of sPLA2-IIA on cell proliferation, we have performed immunofluorescence staining of Ki67, a proliferation marker on wholemount of intact epidermal sheets (Fig. 2a). Our results showed increased number of Ki67 positive cells in the outer root sheath and dermal papillae of the hair follicles, suggesting enhanced proliferation in K14-sPLA2-IIA homozygous mice SCIenTIfIC RepOrTS | 7: 11619 | DOI:10.1038/s41598-017-11830-9
2
www.nature.com/scientificreports/
Figure 1. sPLA2-IIA overexpression altered the hair cycle with abnormal hair follicle morphology and hair loss. (a) Phenotypic appearance of WT, hemizygous and homozygous mice at PD21. (b) Images represents hematoxylin and eosin staining (H&E) of skin sections at various postnatal days of WT and K14-sPLA2IIA homozygous mice to study hair follicle cycling. (c) Graphical representation of epidermal thickness measurements are in μm. Data are presented as mean ± SD. **P
OPEN
Received: 14 December 2016 Accepted: 10 August 2017 Published: xx xx xxxx
Secretory phospholipase A2IIA overexpressing mice exhibit cyclic alopecia mediated through aberrant hair shaft differentiation and impaired wound healing response Gopal L. Chovatiya1,2, Rahul M. Sarate1,2, Raghava R. Sunkara1,2, Nilesh P. Gawas1, Vineet Kala1 & Sanjeev K. Waghmare1,2 Secretory phospholipase A2 Group-IIA (sPLA2-IIA) is involved in lipid catabolism and growth promoting activity. sPLA2-IIA is deregulated in many pathological conditions including various cancers. Here, we have studied the role of sPLA2-IIA in the development of cyclic alopecia and wound healing response in relation to complete loss of hair follicle stem cells (HFSCs). Our data showed that overexpression of sPLA2-IIA in homozygous mice results in hyperproliferation and terminal epidermal differentiation followed by hair follicle cycle being halted at anagen like stage. In addition, sPLA2-IIA induced hyperproliferation leads to compl pathological conditions including various cancers. Here ete exhaustion of hair follicle stem cell pool at PD28 (Postnatal day). Importantly, sPLA2-IIA overexpression affects the hair shaft differentiation leading to development of cyclic alopecia. Molecular investigation study showed aberrant expression of Sox21, Msx2 and signalling modulators necessary for proper differentiation of inner root sheath (IRS) and hair shaft formation. Further, full-thickness skin wounding on dorsal skin of K14-sPLA2-IIA homozygous mice displayed impaired initial healing response. Our results showed the involvement of sPLA2-IIA in regulation of matrix cells differentiation, hair shaft formation and complete loss of HFSCs mediated impaired wound healing response. These novel functions of sPLA2-IIA may have clinical implications in alopecia, cancer development and ageing. Skin constantly renews itself throughout the adult life that acts as a protective barrier against pathogens, radiation etc.1. Adult skin is mainly composed of epidermis, dermis and hypodermis. Epidermal components mainly include interfollicular epidermis (IFE), hair follicle and sebaceous gland2. Dermis and hypodermis are mainly composed of collagen, elastic fibers and extrafibrillar matrix with various cell types including fibroblasts, adipose cells and macrophages. During embryogenesis, hair follicle is formed as an appendage of the epidermis by condensation of specialized mesenchymal cells (dermal papilla) in the dermis. The basal layer cells overlying mesenchymal cells get stimulated and subsequently form placode at E14.5 that proliferate and grow downward as a mature hair follicle at E16.5-E17.53. The hair follicle cycle comprises of distinct stages such as telogen (resting phase), anagen (growth phase) and catagen (regression phase)4. Initially, both the pulse-chase studies carried out using tritiated thymidine (3H) and BrdU showed the presence of infrequently dividing cells in the bulge of hair follicle5. Subsequently, pTre-H2BGFP/K5tTa (Tet off) double transgenic mice study showed that the hair follicle stem cells are highly dynamic, which divide infrequently and undergo random chromosome segregation to 1
Stem Cell Biology Group, Waghmare Lab, Cancer Research Institute, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, 410210, MH, India. 2Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai, 400085, India. Gopal L. Chovatiya and Rahul M. Sarate contributed equally to this work. Correspondence and requests for materials should be addressed to S.K.W. (email: [email protected])
SCIenTIfIC RepOrTS | 7: 11619 | DOI:10.1038/s41598-017-11830-9
1
www.nature.com/scientificreports/ maintain tissue homeostasis6–8. During follicle regeneration, the dermal papilla at the base of hair follicle provides initiatory signalling cues generated from the mesenchymal niche9. The cyclic growth of hair follicle is coordinated by the stem cells residing at the base of the bulge, which proliferate and migrate to provide progeny required for the hair-follicle regeneration and hair growth10. Further, the temporal activity of hair follicle stem cells is strictly dependent on interplay between mesenchymal niche and bulge, which is governed by secretion of various signalling modulators such as Wnt, BMP, Shh, and FGF11. In particular, Wnt signalling is necessary for the hair follicle morphogenesis and required for stem cell proliferation and differentiation during hair follicle regeneration12–14. Additionally, EGF induced EGFR signalling is indispensable for the initiation of hair growth15. The EGF signalling modulator, secretory phospholipase A2 group IIA (sPLA2-IIA) is also known as enhancing factor (EF), which expressed by paneth cells in the small intestine16, 17. sPLA2-IIA has two independent activities, catalytic and non-catalytic (enhancing) and both these activities rely on the two different domains of this enzyme18. Also, various studies have reported the expression of sPLA2-IIA in mouse epidermis19, 20. Moreover, sPLA2-IIA has been implicated in various forms of cancer such as intestinal, colorectal, prostrate21 and overexpression of sPLA2-IIA in mice epidermis showed increased susceptibility towards chemical carcinogenesis22. We have recently reported that sPLA2-IIA enhances the expression of Hb-EGF, EPGN and downstream c-Jun and Fos-B23. However, the molecular insight, if sPLA2-IIA regulates the hair shaft differentiation and the development of alopecia is still unknown. Secondly, do cells of other epidermal components form hair in the complete absence of hair follicle stem cells? Thirdly, whether wound healing process is impaired in the absence of HFSCs is yet to be determined. Notably, deregulation of various signalling modulators perturbs stem cells maintenance that may results in development and progression of cyclic alopecia. Impaired EGFR signalling by dominant negative mutant of epidermal growth factor receptor in the epidermis prevents the progression of the hair cycle to catagen stage and causes severe alopecia24. Further, knockout of Sox21 in mice alters differentiation of cuticle layer leading to development of cyclic alopecia25. Also, epidermal ablation of Smad4 resulted in hyperplasia of interfollicular epidermis (IFE) and sebaceous glands (SGs), that leads to exhaustion of the SC niche and progressive hair loss26. Expression of noggin in epidermis resulted in upregulated Wnt signalling with epidermal hyperplasia, progressive hair loss, and formation of trichofolliculoma-like tumors27. Msx2 deficiency exhibits abnormal structure of hair shafts and cycles of hair loss and regrowth28. Targeted disruption of Orai1 gene in mouse showed sporadic hair loss while inducible deletion of cnB1 gene in mice resulted in altered hair follicle structure and its mesenchyme adhesion, that causes cyclic alopecia29, 30. However, the molecular mechanism involved in cyclic alopecia is yet to be discovered. In this report, we studied the effect of K14-sPLA2-IIA expression in homozygous mice on alopecia and wound healing. To our knowledge, for the first time our findings on K14-sPLA2-IIA homozygous mice showed hair loss that is associated with an increased proliferation and differentiation of hair follicle stem cells, which led to exhaustion of hair follicle stem cells and development of cyclic alopecia at an early age. Additionally, K14-sPLA2-IIA homozygous mice showed impaired healing response during full thickness epidermal wounding.
Results
Altered hair follicle development and hair cycling in K14-sPLA2-IIA homozygous mice. To
check the expression pattern of sPLA2-IIA in mice epidermis, we performed the IHC staining of sPLA2-IIA on skin sections of wild type FVB mice at various postnatal ages. Our data showed that sPLA2-IIA expresses in basal layer, suprabasal layer and outer root sheath of hair follicle during morphogenesis, first hair cycle and one year old age (Supplementary Fig. S1). K14-sPLA2-IIA homozygous mice exhibited visible phenotypic growth abnormalities and are significantly smaller than the control littermates (Fig. 1a). Further, to confirm the nutritional status of the K14-sPLA2-IIA homozygous mice, we have quantified various nutritional parameters from the serum of the K14-sPLA2-IIA homozygous mice. Our data showed that there are no significant alterations in serum components such as serum albumin, Vitamin D 25-OH, Triglyceride, Sodium, Chloride, and marginal increase was observed in total protein, Vitamin B12, Calcium and Potassium (Supplementary Fig. S4). However, we have observed reduced serum glucose level after eight hours fasting in the K14-sPLA2-IIA homozygous mice (Supplementary Fig. S4). These results demonstrate that there are no significant alterations in the nutritional parameters of the K14-sPLA2-IIA homozygous mice. These K14-sPLA2-IIA homozygous mice showed progressive hair loss during hair follicle morphogenesis periods (PD15 and PD19). Further, haematoxylin and eosin staining (H&E) on the dorsal skin sections was performed during various postnatal days (PD15, 19, 21, 25, 28, 35, 41 and 49) (Fig. 1b). Our histological data revealed interfollicular epidermal cyst formation and abnormal thickening of the interfollicular epidermis (IFE) (Fig. 1c) as compared to wild type control littermate. Further, hair follicle cycling analysis on the dorsal skin at various postnatal days showed hair follicle is halted at anagen like stage in K14-sPLA2-IIA homozygous mice (Fig. 1d). This is due to the fact that we have not observed telogen at any postnatal day (PD15, PD17, PD21, PD25, PD28, PD30, PD35, PD41, PD45 and PD49). The change in morphology of the hair follicle is due to abnormal development of epidermal compartments. Further, we observed increased activation of β-catenin in K14-sPLA2-IIA homozygous mice skin as compared to hemizygous and wild type control littermate (Supplementary Fig. S2). These data indicate that the hair follicles of K14-sPLA2IIA homozygous mice failed to progress into regression phase (Catagen) of the hair follicle cycle. Thus, epidermal overexpression of sPLA2-IIA results in morphological abnormalities of hair follicle with hair loss.
Abnormal organization of epidermal components and stratification of epidermis.
Morphological abnormalities of the hair follicle led us to investigate if there is any effect on proliferation and differentiation in different epidermal compartments. To evaluate the effect of sPLA2-IIA on cell proliferation, we have performed immunofluorescence staining of Ki67, a proliferation marker on wholemount of intact epidermal sheets (Fig. 2a). Our results showed increased number of Ki67 positive cells in the outer root sheath and dermal papillae of the hair follicles, suggesting enhanced proliferation in K14-sPLA2-IIA homozygous mice SCIenTIfIC RepOrTS | 7: 11619 | DOI:10.1038/s41598-017-11830-9
2
www.nature.com/scientificreports/
Figure 1. sPLA2-IIA overexpression altered the hair cycle with abnormal hair follicle morphology and hair loss. (a) Phenotypic appearance of WT, hemizygous and homozygous mice at PD21. (b) Images represents hematoxylin and eosin staining (H&E) of skin sections at various postnatal days of WT and K14-sPLA2IIA homozygous mice to study hair follicle cycling. (c) Graphical representation of epidermal thickness measurements are in μm. Data are presented as mean ± SD. **P
